The present invention relates to a human Type 2 RNase H which has now been cloned, expressed and purified to electrophoretic homogeneity. The present invention further relates to oligonucleotide compositions that may serve as substrates for human RNase H1 or human Type 2 RNase H.
RNase H hydrolyzes RNA in RNA-DNA hybrids. This enzyme was first identified in calf thymus but has subsequently been described in a variety of organisms (Stein, H. and Hausen, P., Science, 1969, 166, 393-395; Hausen, P. and Stein, H., Eur. J. Biochem., 1970, 14, 278-283). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (Itaya, M. and Kondo K. Nucleic Acids Res., 1991, 19, 4443-4449; Itaya et al., Mol. Gen. Genet., 1991 227, 438-445; Kanaya, S., and Itaya, M., J. Biol. Chem., 1992, 267, 10184-10192; Busen, W., J. Biol. Chem., 1980, 255, 9434-9443; Rong, Y. W. and Carl, P. L., 1990, Biochemistry 29, 383-389; Eder et al., Biochimie, 1993 75, 123-126). Although RNase Hs constitute a family of proteins of varying molecular weight, nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNase Hs studied to date function as endonucleases, exhibiting limited sequence specificity and requiring divalent cations (e.g., Mg2+, Mn2+) to produce cleavage products with 5xe2x80x2 phosphate and 3xe2x80x2 hydroxyl termini (Crouch, R. J., and Dirksen, M. L., Nuclease, Linn, S, M., and Roberts, R. J., Eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y. 1982, 211-241).
In addition to playing a natural role in DNA replication, RNase H has also been shown to be capable of cleaving the RNA component of certain oligonucleotide-RNA duplexes. While many mechanisms have been proposed for oligonucleotide mediated destabilization of target RNAs, the primary mechanism by which antisense oligonucleotides are believed to cause a reduction in target RNA levels is through this RNase H action. Monia et al., J. Biol. Chem., 1993, 266:13, 14514-14522. In vitro assays have demonstrated that oligonucleotides that are not substrates for RNase H can inhibit protein translation (Blake et al., Biochemistry, 1985, 24, 6139-4145) and that oligonucleotides inhibit protein translation in rabbit reticulocyte extracts that exhibit low RNase H activity. However, more efficient inhibition was found in systems that supported RNase H activity (Walder, R. Y. and Walder, J. A., Proc. Nat""l Acad. Sci. USA, 1988, 85, 5011-25 5015; Gagnor et al., Nucleic Acid Res., 1987, 15, 10419-10436; Cazenave et al., Nucleic Acid Res., 1989, 17, 4255-4273; and Dash et al., Proc. Nat""l Acad. Sci. USA, 1987, 84, 7896-7900.
Oligonucleotides commonly described as xe2x80x9cantisense oligonucleotidesxe2x80x9d comprise nucleotide sequences sufficient in identity and number to effect specific hybridization with a particular nucleic acid. This nucleic acid or the protein(s) it encodes is generally referred to as the xe2x80x9ctarget.xe2x80x9d Oligonucleotides are generally designed to bind either directly to mRNA transcribed from, or to a selected DNA portion of, a preselected gene target, thereby modulating the amount of protein translated from the mRNA or the amount of mRNA transcribed from the gene, respectively. Antisense oligonucleotides may be used as research tools, diagnostic aids, and therapeutic agents.
xe2x80x9cTargetingxe2x80x9d an oligonucleotide to the associated nucleic acid, in the context of this invention, also refers to a multistep process which usually begins with the identification of the nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a foreign nucleic acid from an infectious agent. The targeting process also includes determination of a site or sites within this gene for the oligonucleotide interaction to occur such that the desired effect, either detection or modulation of expression of the protein, will result.
RNase H1 from E.coli is the best-characterized member of the RNase H family. The 3-dimensional structure of E.coli RNase HI has been determined by x-ray crystallography, and the key amino acids involved in binding and catalysis have been identified by site-directed mutagenesis (Nakamura et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 11535-11539; Katayanagi et al., Nature, 1990, 347, 306-309; Yang et al., Science, 1990, 249, 1398-1405; Kanaya et al., J. Biol. Chem., 1991, 266, 11621-11627). The enzyme has two distinct structural domains. The major domain consists of four xcex1 helices and one large xcex2 sheet composed of three antiparallel xcex2 strands. The Mg2+ binding site is located on the xcex2 sheet and consists of three amino acids, Asp-10, Glu-48, and Gly-11 (Katayanagi et al., Proteins: Struct., Funct., Genet., 1993, 17, 337-346). This structural motif of the Mg2+ binding site surrounded by xcex2 strands is similar to that in DNase I (Suck, D., and Oefner, C., Nature, 1986, 321, 620-625). The minor domain is believed to constitute the predominant binding region of the enzyme and is composed of an xcex1 helix terminating with a loop. The loop region is composed of a cluster of positively charged amino acids that are believed to bind electrostatistically to the minor groove of the DNA/RNA heteroduplex substrate. Although the conformation of the RNA/DNA substrate can vary, from A-form to B-form depending on the sequence composition, in general RNA/DNA heteroduplexes adopt an A-like geometry (Pardi et al., Biochemistry, 1981, 20, 3986-3996; Hall, K. B., and Mclaughlin, L. W., Biochemistry, 1991, 30, 10606-10613; Lane et al., Eur. J. Biochem., 1993, 215, 297-306). The entire binding interaction appears to comprise a single helical turn of the substrate duplex. Recently the binding characteristics, substrate requirements, cleavage products and effects of various chemical modifications of the substrates on the kinetic characteristics of E.coli RNase HI have been studied in more detail (Crooke, S. T. et al., Biochem. J., 1995, 312, 599-608; Lima, W. F. and Crooke, S. T., Biochemistry, 1997, 36, 390-398; Lima, W. F. et al., J. Biol. Chem., 1997, 272, 18191-18199; Tidd, D. M. and Worenius, H. M., Br. J. Cancer, 1989, 60, 343; Tidd, D. M. et al., Anti-Cancer Drug Des., 1988, 3, 117.
In addition to RNase HI, a second E.coli RNase H, RNase HII has been cloned and characterized (Itaya, M., Proc. Natl. Acad. Sci. USA, 1990, 87, 8587-8591). It is comprised of 213 amino acids while RNase HI is 155 amino acids long. E. coli RNase HIM displays only 17% homology with E.coli RNase HI. An RNase H cloned from S. typhimurium differed from E.coli RNase HI in only 11 positions and was 155 amino acids in length (Itaya, M. and Kondo K., Nucleic Acids Res., 1991, 19, 4443-4449; Itaya et al., Mol. Gen. Genet., 1991, 227, 438-445). An enzyme cloned from S. cerevisae was 30% homologous to E.coli RNase HI (Itaya, M. and Kondo K., Nucleic Acids Res., 1991, 19, 4443-4449; Itaya et al., Mol. Gen. Genet., 991, 227, 438-445). Thus, to date, no enzyme cloned from a species other than E. coli has displayed substantial homology to E.coli RNase HII.
Proteins that display RNase H activity have also been cloned and purified from a number of viruses, other bacteria and yeast (Wintersberger, U. Pharmac. Ther., 1990, 48, 259-280). In many cases, proteins with RNase H activity appear to be fusion proteins in which RNase H is fused to the amino or carboxy end of another enzyme, often a DNA or RNA polymerase. The RNase H domain has been consistently found to be highly homologous to E.coli RNase HI, but because the other domains vary substantially, the molecular weights and other characteristics of the fusion proteins vary widely.
In higher eukaryotes two classes of RNase H have been defined based on differences in molecular weight, effects of divalent cations, sensitivity to sulfhydryl agents and immunological cross-reactivity (Busen et al., Eur. J. Biochem., 1977, 74, 203-208). RNase H Type 1 enzymes are reported to have molecular weights in the 68-90 kDa range, be activated by either Mn2+ or Mg2+ and be insensitive to sulfhydryl agents. In contrast, RNase H Type 2 enzymes have been reported to have molecular weights ranging from 31-45 kDa, to require Mg2+ to be highly sensitive to sulfhydryl agents and to be inhibited by Mn2+ (Busen, W., and Hausen, P., Eur. J. Biochem., 1975, 52, 179-190; Kane, C. M., Biochemistry, 1988, 27, 3187-3196; Busen, W., J. Biol. Chem., 1982, 257, 7106-7108.).
An enzyme with Type 2 RNase H characteristics has been purified to near homogeneity from human placenta (Frank et al., Nucleic Acids Res., 1994, 22, 5247-5254). This protein has a molecular weight of approximately 33 kDa and is active in a pH range of 6.5-10, with a pH optimum of 8.5-9. The enzyme requires Mg2+ and is inhibited by Mn2+ and n-ethyl maleimide. The products of cleavage reactions have 3xe2x80x2 hydroxyl and 5xe2x80x2 phosphate termini.
Despite the substantial information about members of the RNase family and the cloning of a number of viral, prokaryotic and yeast genes with RNase H activity, until now, no mammalian RNase H had been cloned. This has hampered efforts to understand the structure of the enzyme(s), their distribution and the functions they may serve.
In the present invention, a cDNA of human RNase H with Type 2 characteristics and the protein expressed thereby are provided.
The present invention provides oligonucleotides that can serve as substrates for human RNase H1. These oligonucleotides are mixed sequence oligonucleotides comprising at least two portions wherein a first portion is capable of supporting human RNase H1 cleavage of a complementary target RNA and a further portion which is not capable of supporting such human RNase H1 cleavage.
The present invention provides a mixed sequence oligonucleotide comprising at least 12 nucleotides and having a 3xe2x80x2 end and a 5xe2x80x2 end, said oligonucleotide being divided into a first portion and a further portion,
said first portion being capable of supporting cleavage of a complementary target RNA by human RNase H1 polypeptide,
said further portion being incapable of supporting said RNase H cleavage;
wherein said first portion comprises at least 6 nucleotides and is positioned in said oligonucleotide such that at least one of said 6 nucleotides is 8 to 12 nucleotides from the 3xe2x80x2 end of said oligonucleotide.
In a preferred embodiment the oligonucleotide comprises at least one CA nucleotide sequence. In another embodiment the first portion of the mixed sequence oligonucleotide of the present invention comprises nucleotides having a B-form conformational geometry. In a further embodiment each of the nucleotides of the first portion of the oligonucleotide are 2xe2x80x2-deoxyribonucleotides. In a still further embodiment each of the nucleotides of the first portion of the oligonucleotide is a 2xe2x80x2-F arabinonucleotide or a 2xe2x80x2-OH arabinonucleotide. In yet another embodiment the nucleotides of the first portion are joined together in a continuous sequence by phosphate, phosphorothioate, phosphorodithioate or boranophosphate linkages. In yet a further embodiment all of the nucleotides of the further portion of the oligonucleotide are joined together in a continuous sequence by 3xe2x80x2-5xe2x80x2 phosphodiester, 2xe2x80x2-5xe2x80x2 phosphodiester, phosphorothioate, Sp phosphorothioate, Rp phosphorothioate, phosphorodithioate, 3xe2x80x2-deoxy-3xe2x80x2-amino phosphoroamidate, 3xe2x80x2-methylenephosphonate, methylene(methylimino), dimethylhydrazino, amide 3, amide 4 or boranophosphate linkages.
Yet another object of the present invention is to provide methods for identifying agents which modulate activity and/or levels of human RNase H1. In accordance with this aspect, the polynucleotides and polypeptides of the present invention are useful for research, biological and clinical purposes. For example, the polynucleotides and polypeptides are useful in defining the interaction of human RNase H1 and antisense oligonucleotides and identifying means for enhancing this interaction so that antisense oligonucleotides are more effective at inhibiting their target mRNA.
Yet another object of the present invention is to provide a method of prognosticating efficacy of antisense therapy of a selected disease which comprises measuring the level or activity of human RNase H in a target cell of the antisense therapy. Similarly, oligonucleotides can be screened to identify those oligonucleotides which are effective antisense agents by measuring binding of the oligonucleotide to the human RNase H1.